Plants contain rhamnogalacturonans, i.e. polysaccharides with more or less regularly alternating rhamnose and galacturonic acid residues in the backbone. The rhamnogalacturonans may have mono, oligo, and polysaccharide side-branches. Rhamnogalacturonans are part of the pectin-polymers, a major component of the plant cell walls.
Most pectin-polymers are composed of smooth regions, i.e. linear homogalacturonan, and hairy (ramified) regions. The hairy regions consist of a rhamnogalacturonan backbone with side-branches of varying length. The side-branches includes monosaccharides like xylose, galactose and arabinose, and oligo and polysaccharides like araban, galactan and arabinogalactan. Further the rhamnogalacturonan backbone is methylated and acetylated. The composition of the very complex structure of the hairy regions vary according to the source of the plant cell wall, cf Fry (1988), Schols et al. (1990b), O'Neill et al. (1990), Voragen and Schols (1992) and Carpita and Gibeaut (1993).
The enzymatic liquefaction and degradation of plant materials (e.g. fruits, vegetables, cereals, oil fruits and seeds) by technical processes involves combinations of pectolytic, cellulolytic and proteolytic enzyme preparations. However the hairy regions of pectin cause problems in such processes, because they are resistant to degradation of most technical enzyme preparations. A more extensive degradation of hairy regions is desired in many processes in order to improve the liquefaction and degradation of the plant material. For instance, an extensive degradation is important in processing of clear liquids and in processing of viscous plant cell wall containing material where a viscosity reduction is otherwise difficult to obtain. Furthermore, a more specific enzymatic degradation of the hairy regions is desirable for e.g. production of cloudy liquids, purification of pectin and soluble dietary fibres.
For these processes a degradation of the backbone of pectin hairy regions is of major importance. The degradation of the backbone of the hairy regions is performed by enzymes designated rhamnogalacturonases (RGases). RGases are believed to hydrolyse the bond between rhamnose and galacturonic acid. In order to facilitate the activity of RGases it may be desirable to reduce the degree of acetylation of the backbone, e.g. by use of the enzyme rhamnogalacturonan acetyl esterase (cf Searle-van Leeuwen et al., 1992). Furthermore, a reduced degree of branching of parts of the hairy regions may be desirable. The reduced degree of branching may be obtained by enzymes which attacks the side-branches, like galactanases, arabinanases, beta-galactosidases, alpha-arabinosidases and beta-xylosidases.
The isolation and purification of a RG'ase from Aspergillus aculeatus is described by Schols et al. (1990a).
WO 92/19728, the contents of which are incorporated by reference herein, discloses partial amino acid sequences of different RGases isolated from the Aspergillus sp. A. aculeatus and A. japonicus and from Irpex lacteus. EP 570 075 discloses an Aspergillus RGase gene and the construction of recombinant Aspergillus strains which overexpress RGase.
The RGase described by Schols et al., (1990a) has been found to have a similar degradation pattern to the A. aculeatus RGase described in WO 92/19728 and has been found to be immunologically cross-reactive with said RGase. RGase of this type is termed RGase II in the following disclosure.
Furthermore, in an article of Colquhoun (1990), the composition of a mixture of oligosaccharides obtained by enzymatic degradation of the modified hairy (ramified) regions of apple pectin with RGase II is described. It is shown that RGase II hydrolyses in the rhamnogalacturonan backbone leaving rhamnose as the non-reducing end in the degradation products.
Furthermore, RGase II type activity isolated from Trametes sanguinea on protopectin extracted from sugar beet has been reported recently by Sakamoto et al., 1993.
It has been shown that A. aculeatus RGase II exhibits optimum activity in the pH range of 3-4, which is lower than the pH of most plant materials and lower than desired pH in most industrial processes. The RGase disclosed in EP 570 075 and the Trametes sanguinea RGase mentioned above are stated to be used at pH 5.0.
High activity of RGase II has only been demonstrated on hairy regions from a limited number of plants, there is no reports of a significant activity of RGase II on hairy regions from soy and beets.
Especially for the industries dealing with modifications of plant cell walls for e.g. human nutrition and for animal feed (e.g. liquefaction of fruits, vegetables, cereals, oil fruits and seeds), it is important to provide a variety of different RGases (in respect to mode of action, pH and temperature range) in order to be able to exploit the desirable actions of RGases under widely varying technical process conditions. In particular there is a need for rhamnogalacturonase which is active at a higher pH than RGase II and which is active on plant materials for which RGase II has only limited activity.